Peroxisome proliferator-activated receptor α (PPARα) is a member of the nuclear/steroid receptor gene superfamily that plays an essential role in fatty acid metabolism. PPARα modulates the expression of genes encoding peroxisomal fatty acid β-oxidation enzymes and microsomal fatty acid hydroxylases CYP4As. We have previously reported that the obligate Eucalyptus feeder koala (Phascolarctos cinereus) exhibits a higher hepatic CYP4A activity and an absence of peroxisomal palmitoyl-CoA oxidation as compared to non-Eucalyptus feeders human, rat or wallaby. Here we describe the cloning, expression and molecular characterisation of koala hepatic PPARα. A full-length PPARα cDNA of size 1515 bp was cloned by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The koala PPARα cDNA encodes a protein of 468 amino acids. Transfection of the koala PPARα cDNA into Cos-7 cells resulted in the expression of a protein recognised by a rabbit anti-human PPARα polyclonal antibody. PPARα immunoreactive bands of the same molecular mass were detected in nuclear extracts of koala livers. The results of this study demonstrate the presence of koala hepatic PPARα which shares several common features with other published PPARαs; however, it exhibits important differences in both the DNA and ligand binding domains.